Chemical synthesis of peptides is well established. In principle, two different methods are distinguished: the synthesis in solution, which is often very time consuming and therefore not useful for scientific research, and the synthesis on a solid support, which allows a fast optimization of reaction cycles. The protocols available for solid phase peptide synthesis (SPPS) are based on the Merrifield technique (Merrifield, R. B., J. Amer. Chem. Soc. 85, 1963, 2149) for synthesizing peptides with defined sequences on an insoluble solid phase. The general principle of SPPS is based on the repetition of cycles of coupling-deprotection: the free N-terminal amine of a peptide attached on a solid phase by its carboxyl end is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached.
However, present SPPS methods produce, in addition to the target compounds (the mature peptides), a relatively large number of impurities, and particularly a large amount of immature peptides. Purification of peptides derived from solid-phase peptide synthesis (SPPS) hence requires the removal of deleted peptides (ie peptides lacking one or several amino acid residues) resulting from incomplete coupling/deprotection steps and, in a much lesser extent, other peptide co-products from racemisation or side-chain rearrangement, and of various chemical substances introduced during the deprotection or cleavage stages of an SPPS procedure. In particular, the more the peptides to be synthesized are long, the more the number of impurities and in particular the number of deleted peptides is. Therefore, an important objective of a SPPS method is to recover the target peptide alone from impurities with high speed and high yield.
It has thus been proposed to perform a capping by acetic anhydride after every coupling reaction to terminate further elongation of peptide chains of a non-target sequence and to avoid further production of deleted peptides and obtain truncated peptides. After the coupling of the final amino acid, only the peptide having a complete amino acid sequence will have an amino group at its N-terminus: this amino group can be used to purify the target peptide.
Several reports on peptide purification methods using the N-terminus amino group have been published. However, none of these methods has been able to achieve effective one-step separation; instead complicated separation processes are required.
Another method has been developed in which the target peptide alone is elongated with two extra residues (cysteine-methionine) at its N-terminus, then reacted with a solid support derivatized with a phenyl-mercury group taking advantage of the selective binding of the SH group of the cysteine. Subsequent to the separation, the methionine-peptide amide bond is selectively cleaved by BrCN to yield the target peptide (D. E. Krieger et al., Proc. Natl. Acad. Sci. U.S.A., 73, 3160 (1976)). However, this method has a limitation of being not applicable to peptides containing methionine or cysteine.
Still another method has been disclosed in which the target peptide is covalently linked to a solid support through a SH group (U.S. Pat. No. 5,648,462 and No. 5,994,588). However, this method has a limitation of being not applicable to peptides containing cysteine.
There is thus a need for further methods for purifying the peptides produced by SPPS, said methods being applicable to any type of peptides and being easy to carry out.